Recently, the estimation of the measurement uncertainty has become a significant issue in the quality control of forensic drug testing. In the present study, the uncertainty of the measurement was calculated for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide conjugate (THC-COOH-glu) in urine using liquid chromatography-tandem mass spectrometry. The procedure was based on liquid-liquid extraction of a volume of urine (800 µL) with ethyl acetate. The sources of uncertainty were identified and classified into four major categories as follows: standard preparation, calibration curve, method precision and bias. The overall contribution of combined standard uncertainty on THC-COOH increased in the order of standard preparation (0.9%), method precision (10.4%), calibration curve (30.3%) and bias (58.4%) and, while calibration curve (53.0%) and bias (40.4%) gave the bigger contributions to the combined standard uncertainty for THC-COOH-glu than method precision and standard preparation, which accounted for 6.3 and 0.3%, respectively. The reliability of a measurement was expressed by stating the expanded uncertainty of the measurement result at 95% confidence level. The concentrations of THC-COOH and THC-COOH-glu in the urine sample with their expanded uncertainties were 10.20 ± 1.14 ng/mL and 25.42 ± 5.01 ng/mL, respectively.
Cannabis, or marijuana, the most commonly used illicit drug in the world, has been shown to be responsible for suppressing the production and secretion of androgens, particularly testosterone. However, despite such findings in animals, the chronic effects of marijuana use on human endocrine systems have proved to be inconsistent. Here, we investigated the reference ranges of urinary levels of testosterone (T) and epitestosterone (E) as well as their metabolic ratio of T/E in a Korean male population (n=337), which would enable an evaluation of abnormal changes in steroid metabolism induced by habitually administered cannabis. The T/E ratio was significantly decreased in the marijuana group (n=18), while the urinary testosterone concentrations were also tended to decrease. This study is the first to provide data for the reference values of two urinary androgens and T/E values among control Korean males, and, furthermore, suggests that the T/E ratio, though not testosterone levels, might be used to understand the suppression of human male gonadal function affected by smoking marijuana.
A liquid chromatography-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) was developed and validated for the direct determination of 11-nor-D 9 -tetrahydrocannabinol-9-carboxylic acid (THCCOOH) and its glucuronide (THCCOOH-glu) in urine. The deuterium-labeled compounds were used as internal standards to quantify the analytes. After a simple liquid-liquid extraction with ethyl acetate at pH 4, the dried extracts were reconstituted in the mobile phase followed by injection onto the LC-ESI-MS/MS system. The mobile phase was composed of 0.2% formic acid and 2 mM ammonium formate in water and acetonitrile. A reversed phase column (C 8 , 150 mm  2.1 mm i.d., 3.5 mm) was used as the analytical column. The limit of detection and lower limit of quantification values were 0.08 and 0.3 ng mL À1 for THCCOOH and 0.4 and 1.5 ng mL À1 for THCCOOH-glu, respectively. The calibration curve was linear over the concentration range of 0.3-100 ng mL À1 for THCCOOH and 1.5-100 ng mL À1 for THCCOOH-glu with the coefficients of determination (r 2 ) above 0.9955. The intra-and inter-day precisions (% CV) were within 16.6% and 18.3%, respectively. The intra-day accuracies (% bias) ranged from À18.4 to 1.5% while inter-day accuracies were À5.9 to 1.8%. The validated method was successfully applied for the determination of THCCOOH and THCCOOH-glu in urine samples from marijuana users.
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